A variety of novel techniques for analysis of nucleic acids best utilize relatively short, single-stranded analytes. Many such techniques are based on the hybridization of analyte nucleic acids to miniaturized arrays of short (&lt;25 nucleotides), single-stranded DNA probes (Ramsay, Nat. Biotechnol. 16:40 (1998); Marshall et al, J. Nat. Biotechnol. 16:27 (1998)). Samples of analyte DNA often arise by PCR amplification from a biological sample, range from hundreds to thousands of nucleotides and are obtained as double-stranded molecules. As hybridization requires single-stranded nucleic acids, various means have been devised to render amplification products single-stranded.
The stability of duplex nucleic acid is directly proportional to molecular length. Thus, it can be difficult to form single-stranded analytes from long, double-stranded amplicons. Further, it has been observed in some instances that the performance of various nucleic acid analysis techniques, such as the above-referenced array-based methods, is superior with shorter analyte nucleic acids. This has created a demand for methods of fragmenting analyte nucleic acid or amplification products thereof.
Available fragmentation methods include limited restriction digestion of the amplicon, as well as the incorporation of uridine during amplification followed by backbone cleavage using uracil-N-glycosylase. These methods, however, have drawbacks related to their a sequence bias and the additional processing steps needed to remove reagents/byproducts. Accordingly, there is a clear need for a nucleoside analog that can be enzymatically incorporated at random within a nucleic acid chain and subsequently activated for cleavage of the backbone.
A number of workers have developed analogs of the heterocyclic bases in nucleic acids for various purposes. For example, Bergstrom et al have advanced nitropyrrole and nitroindole as heterocycles that are accepted opposite any of the natural bases in double-stranded nucleic acid (Bergstrom et al, Nucleic Acids Res. 25:1935 (1997); Bergstrom et al, J. Am. Chem. Soc. 117:1201 (1995)). Such "universal" bases have uses including simplified syntheses of short oligonucleotides that are suitable for use as probes for hybridization, and as primers for DNA sequencing and nucleic acid amplification. On the other hand, Kool et al have shown that difluorotoluene specifically substitutes for the base thymidine (T) in duplex DNA with only a small loss in hybrid stability (Schweitzer et al, J. Org. Chem. 59:7238 (1994)). Further, difluorotoluene is accepted by DNA polymerases either in the template strand or as an incoming triphosphate (Moran et al, Proc. Natl. Acad. Sci. USA 94:10506 (1997); Moran et al, J. Am. Chem. Soc. 119:2056 (1997); Liu et al, Chem. Biol. 4:919 (1997)). As a result, high fidelity complementation of the unnatural base with a natural adenine (A) occurs. Finally, Ordoukhanian et al (J. Am. Chem. Soc. 117:9570 (1995)) have developed unnatural base analogs that can be cleaved into two portions (and thereby cleave the backbone of the nucleic acid) by irradiation. These compounds do not resemble natural nucleosides and are not accepted by nucleic acid modifying enzymes. Thus, they must be incorporated into oligonucleotides by chemical synthesis.
The present invention provides a nucleoside analog that can be enzymatically incorporated into a nucleic acid chain and then activated for cleavage of that chain.